The aim of this proposal is to develop an atomic-level understanding of the mechanism of lactose/H+ symport by the lactose permease of Escherichia coli (LacY), an important model for the Major Facilitator Superfamily (MFS), the largest family of membrane transport proteins. The MFS contains many members that have clinical significance such as the vesicular monoamine transporter, as well as the GLUTs, which facilitate glucose transport into many different cells in the body. Like channels and ABC transporters, symporters are also highly relevant to human health (e.g. transport across epithelia, synaptic function) and disease (e.g. depression, epilepsy, diabetes, multidrug resistance). Also notable, at least two of the most widely used pharmaceuticals in the world [serotonin selective reuptake inhibitors (SSRIs) and gastric proton pump inhibitors (PPIs)] are targeted to membrane transport proteins. Also important, the innovative methods developed by the PI to study LacY have been applied directly to such important human membrane proteins as GLUT1 and G-Protein-Coupled Receptors (GPCRs). However, despite a number of structures of MFS members, including 7 of LacY, the mechanism of this dynamic protein is not completely understood. It has been demonstrated that sugar binding to highly dynamic protonated LacY triggers a global conformational change in which sugar- and H+-binding sites gain alternating access to either side of the membrane. Sugar binding and dissociation drive this conformational change through an induced-fit mechanism, while the proton electrochemical gradient enhances the rate of deprotonation. Therefore, LacY behaves much like an enzyme except that the transition state(s) involves the protein rather than the substrate. X-ray structures of LacY inward- and almost occluded outward-facing conformations provide the structural basis for studying the alternating access mechanism. The alternating access mechanism has been documented unequivocally by applying pre-steady state kinetics, as well as multiple biochemical and spectroscopic approaches pioneered in this laboratory, and by using kinetic data obtained in real time for several steps in the transport cycle. Thirty-one Camelid nanobodies now in our possession allow stabilization of LacY in different intermediate states that will provide an in-depth understanding of structural changes underlying the symport mechanism.